Phosphoenolpyruvate carboxykinases, depending on the enzyme origin, preferentially use adenine or guanine nucleotides as substrates. In this work, analyses of the substrate specificity of the Saccharomyces cerevisiae ATP-dependent enzyme have been carried out. Kinetics studies gave relative values of kcat/Km for the nucleoside triphosphate complexes in the order ATP GTP > ITP > UTP > CTP. For the nucleoside diphosphate complexes the order is ADP GDP > IDP UDP > CDP. This shows that the enzyme has a strong preference for ADP (or ATP) over other nucleotides, being this preference about an order of magnitude higher for the diphosphorylated than for the triphosphorylated nucleosides. The calculated binding free energies (kcal mol−1) at 25 °C are 7.39 and 6.51 for ATP and ADP, respectively. These values decrease with the nucleotide structure in the same order than the kinetic specificity. The binding energy for any triphosphorylated nucleoside is more favourable than for the corresponding diphosphorylated compound, showing the relevance of the Pγ for nucleotide binding. Homology models of the adenine and guanine nucleotides in complex with the enzyme show that the base adopts a similar conformation in the diphosphorylated nucleosides while in the triphosphorylated nucleosides the sugar-base torsion angle is 61° for ATP and −53° for GTP. Differences are also noted in the distance between Pβ and Mn2+ at site 1. This distance is almost the same in the ATP, GTP, and UTP complexes, however in the ADP, GDP and UDP complexes it is 2.9, 5.1, and 7 Å, respectively. Experimental data obtained with a Thr463Ala mutant enzyme agree with molecular simulation predictions. The results here presented are discussed in terms of the proposed interactions of the nucleotides with the protein.